Pharmaceuticals

Lung cancer continues to be the leading cause of cancer-related mortality globally, with non-small cell lung cancer (NSCLC) representing the most prevalent subtype. Tumor hypoxia is a characteristic feature of the neoplastic microenvironment in NSCLC, facilitating tumor progression and conferring resistance to oxidative stress through the stabilization of hypoxia-inducible factor-1 alpha (HIF-1). In this study, we investigated the combined anticancer effects of baicalein (Bai), a natural flavonoid, and thermal-cycling stimulation (TCS), a physical treatment that minimizes damage to normal cells, under cobalt (II) chloride (CoCl2)-induced hypoxic conditions in NSCLC. In A549 NSCLC cells, the combination of Bai and TCS significantly decreased cell viability and induced apoptosis, while exhibiting minimal cytotoxicity on IMR-90 normal human lung fibroblast cells. On a mechanistic level, this combined treatment suppressed the expression of HIF-1 and superoxide dismutase 2 (SOD2) proteins, elevated intracellular reactive oxygen species (ROS) levels, and impaired DNA repair capability by downregulating MutT homolog 1 (MTH1) protein expression. Additionally, disruption of mitochondrial membrane potential and increased poly (ADP-ribose) polymerase (PARP) cleavage further confirmed the induction of apoptosis. These findings indicate that combining Bai with TCS offers a promising synergistic approach to treating NSCLC under hypoxic conditions.

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.

Introduction: Nicotine Pouches are increasingly used as a smokeless alternative to cigarettes and other nicotine products, yet their acute cardiovascular effects remain poorly documented. While nicotine's impact on heart rate and electrocardiogram (ECG) parameters is well-documented in smoking, no trials have evaluated these effects specifically for nicotine pouches. Methods: This study is a single-center, double-blind, placebo-controlled, crossover trial which will include 20 healthy adult nicotine users. Participants will undergo three sessions, receiving either a placebo, 6 mg, or 14 mg nicotine pouch in random order. Heart rate obtained by an ECG and various other ECG parameters, vital signs, and subjective symptoms will be measured at baseline, and multiple time points over 30 minutes. Conclusions: This study aims to determine whether nicotine pouches cause acute changes in heart rate, ECG parameters, vital signs, and self-reported symptoms. We hypothesize that higher nicotine pouch does will lead to measurable increases in heart rate and other autonomic effects compared to placebo.

Several potentially potent anticancer drugs have been identified by in vitro evaluation, such as Andrographolide. These compounds show strong anticancer activity in vitro, but struggle to reach effective concentrations in the bloodstream when taken orally because they dissolve poorly in water or break down rapidly in the body. Bioenhancers, which are compounds that have potential to improve drug stability in the body, offer an alternative solution to overcome this limitation. Naringin and Quercetin have been identified as candidate bioenhancers, and have been hypothesized to potentially slow rapid first pass metabolism of poorly bioavailable drugs. Our work focuses on testing Naringin and Quercetin because they are flavonoids with therapeutic potential, due to their anti-inflammatory and antioxidant properties. Data from the hepatic microsomal assays performed on Naringin and Quercetin suggest moderate to proficient periods of stability in the body, with Naringin having 91.86% remaining, while Quercetin had 74.84% remaining. When administered alongside Andrographolide, a drug known to rapidly degrade in the body, Naringin raised its metabolic stability from 38.93% to 80.77% and on the other hand, Quercetin raised Andrographolide metabolic stability from 38.93% to 86.70%. In addition, plasma protein binding assays show the percentage of compounds available at the target site where Naringin was observed to be 49.32% bound and Quercetin found to be 50.14% bound, implying 50.68% of Naringin, and 49.86% of Quercetin available at the target site, respectively. This preliminary study explores whether Quercetin and Naringin could act as bioenhancers by remaining stable and available in plasma and by slowing the metabolism of poorly bioavailable drugs such as Andrographolide.

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

Tobacco use is the leading preventable cause of death worldwide. Anxiety increases the risk for smoking, and smoking in turn increases the risk for anxiety disorders. There is therefore a need to identify interventions that reduce anxiety, in general and in the context of smoking, without producing sedation. Kava (Piper methysticum), a natural product with a long history of indigenous use, has been shown to have anxiolytic and calming effects and reduce nicotine withdrawal. The current study examined whether kava without the hepatotoxic flavokavains A and B (AB-free) could reduce anxiety-like behavior in mice repeatedly treated with nicotine. Male and female C57BL/6NCrl mice received either a control diet or an AB-free kava-supplemented diet and underwent two blocks of nicotine treatments. Mice underwent a first block of five every-other-day injections of nicotine (0.5 mg/kg) or saline, with open field testing after each injection, followed one week later by a nicotine challenge. A second block of injections was given using the same injection schedule, followed by a second challenge one week later, and two weeks afterward mice received a final challenge in a novel open field. During the first treatment block, AB-free kava significantly increased center time overall, an effect most pronounced in saline-treated animals, and increased locomotor activity, while nicotine decreased both measures. During the second challenge, nicotine reduced center time but not locomotor activity, and AB-free kava increased center time in saline-treated animals only. During the final challenge, nicotine reduced both measures, whereas AB-free kava increased center time regardless of nicotine treatment, and kava-treated animals also showed a near-significant increase in center entries. These results suggest that AB-free kava reduces anxiety-like behavior without inducing sedation but does not prevent nicotine-induced suppression of exploratory behavior.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Subtype-selectivity predictions are scored against measured selectivity and judged against an assumed noise ceiling. We asked what an 2-adrenergic benchmark rewards and which controls change its interpretation. Research design and methods: On a frozen benchmark of 586 paired 2A/2C compounds we evaluated Glide SP docking, CNN rescoring, ligand-only fingerprint models, receptor descriptors and pose contacts, with dopamine D3/D2 as comparator, applying five controls: a measured ceiling, a cluster-identity null, a nonselective reference, a same-receptor floor and a trivial-descriptor baseline. Results: Five descriptors from SMILES reached Spearman 0.645, 72% of the measured ceiling, against 0.071 for Glide SP and 0.188 for CNN rescoring; receptor properties and pose contacts reduced to size under control, while a non-size signal of 0.258 survived. Measured rather than propagated noise raised that ceiling from 0.704 to 0.897; cluster identity alone reached R2 0.499 on D3/D2 and none on 2; a nonselective reference received +1.43 to +4.79 kcal/mol where zero is expected; and a same-receptor floor reached 1.77-fold against 1.88-fold across subtypes. Conclusions: Such benchmarks reward molecular size first; a method must exceed 0.645 before its score indicates structural reasoning. The controls are inexpensive; conclusions rest on two receptor pairs, a three-pair floor and static structures.

Osteoarthritis is characterized by inflammation, chondrocyte dysfunction, and progressive cartilage degradation. Boric acid (BA), a physiologically relevant boron compound, has shown anti-inflammatory properties, but its effects on human articular chondrocytes remain unclear. This study investigated whether BA could protect primary human chondrocytes against lipopolysaccharide-induced inflammatory injury. Cell survival, membrane damage, apoptosis, inflammatory mediator production, and expression of genes related to inflammation and extracellular matrix degradation were assessed. BA improved chondrocyte survival and reduced membrane damage and apoptosis following inflammatory stimulation. It also suppressed inflammatory and matrix-degrading gene expression, nitrite production, and the release of proinflammatory mediators. These protective effects were generally more pronounced with the higher treatment dose. Analysis of publicly available human chondrocyte RNA-sequencing datasets provided complementary support for the relevance of several investigated inflammatory and catabolic targets. Overall, these findings demonstrate that BA protects primary human chondrocytes against inflammatory and catabolic injury and support its further investigation as a potential chondroprotective approach in osteoarthritis.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) are aggressive hematological malignancies requiring novel therapeutic strategies. The majority of T-ALL and PTCL tumors display metabolic activation and addiction to endogenous serine/glycine synthesis (SSP), providing opportunities for targeted therapy with the clinically used antidepressant sertraline, inhibiting SSP enzymes SHMT1/2. However, sertraline monotherapy only induces cell cycle arrest and has limited efficacy in suppressing disease progression in vivo. MethodsDrug synergy of sertraline combined with clinically used proteasome inhibitors carfilzomib and bortezomib was evaluated. Drug effects on cell cycle, proliferation and apoptosis were assessed in T-ALL, PTCL and healthy blood cells using flow cytometry assays. Proteomic, lipidomic and metabolic analyses on drug treated T-ALL cells were performed to elucidate the molecular mechanisms underlying drug synergy, followed by validation of changes of interest, metabolic rescues and shRNA-knockdown of SSP enzymes in T-ALL cells. In vivo therapeutic efficacy and immune remodelling were evaluated in an immunocompetent MYCN-overexpressing PTCL mouse model. ResultsSertraline acted synergistically with clinically used proteasome inhibitor carfilzomib to induce cell cycle arrest and apoptosis in T-ALL and PTCL cells with SSP activity, with minimal effects on SSP-inactive T-ALL cells or healthy blood cells. Adding carfilzomib also enhanced the therapeutic efficacy of sertraline in an aggressive MYCN PTCL model. Sertraline rewired cell metabolism towards increased cholesterol uptake and biosynthesis in SSP-active T-ALL cells, and this effect was not obtained by other means of SSP inhibition. In contrast to sertraline, carfilzomib promoted cholesterol efflux. Moreover, carfilzomib reduced total lipid levels, further restricting nutrients in sertraline - carfilzomib treated cells. Additionally, the drug combination impaired mitochondrial respiration and elevated reactive oxygen species (ROS) levels and DNA damage in SSP-active tumor cells, which was rescued by citrate supplementation. Interestingly, these metabolic changes were associated with microenvironmental changes in our mouse model, where the drug combination elevated natural killer T-cells, neutrophils and eosinophils. ConclusionsOur study identifies synergy of sertraline - carfilzomib combination treatment mediated through metabolic impairment and is associated with remodelling of the immune microenvironment. This invites for further clinical investigation of this drug combination as a therapeutic strategy for SSP-active T-cell malignancies.

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8fe6c2org.highwire.dtl.DTLVardef@176cef2org.highwire.dtl.DTLVardef@10c8201org.highwire.dtl.DTLVardef@2b28cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

Cigarette smoke-induced lung inflammation is a central driver of pulmonary diseases. The limited efficacy of current anti-inflammatory agents underscores the need for structurally novel therapeutics with distinct mechanisms. We recently demonstrated that AB-free kava, a flavokavains A/B-depleted formulation from Piper methysticum containing six major kavalactones, effectively suppresses cigarette smoke-induced lung inflammation in mice. This study aims to identify the bioactive constituent(s) and elucidate underlying mechanisms. These kavalactones revealed a clear structure-activity relationship in suppressing lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in macrophages with desmethoxyyangonin (DMY) as the most potent kavalactone whereas dihydrokavain (DHK, a structurally similar analog) with minimal activity. DMY also effectively reduced LPS-induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) production while DHK was ineffective. Mechanistically, DMY, but not DHK, attenuated COX-2 induction and reduced phosphorylation of cAMP response element-binding protein (CREB). Pharmacological inhibition of protein kinase A (PKA) similarly reduced p-CREB, COX-2 and PGE2, supporting a PKA-dependent CREB/COX-2 signaling in mediating PGE2 suppression while these effects were independent of nuclear factor kappa B (NF-{kappa}B) and activator protein 1 (AP-1) signaling. Similar results were observed for DMY and DHK in attenuating cigarette smoke condensate-induced proinflammatory pathways and PGE2 production. Consistently, DMY demonstrated significant in vivo efficacy in suppressing cigarette smoke-induced lung inflammation while DHK was not effective. Interestingly, dihydromethysticin (DHM) demonstrated the greatest in vivo anti-inflammatory efficacy, although it only exhibited moderate in vitro potency, likely due to its superior bioavailability over DMY. Concordantly, cigarette smoke exposure elevated p-CREB and COX-2 expressions in mouse lungs, which were attenuated by AB-free kava and its bioactive kavalactones with the extent of suppression correlating with their in vivo anti-inflammatory efficacy. DHM effectively suppressed LPS-induced neutrophil accumulation in mouse lungs as well. Collectively, these studies identify bioactive kavalactones in AB-free kava that suppress cigarette smoke- and LPS-induced lung inflammation through the modulation of the PKA/CREB/COX-2 signaling axis, providing a foundation for developing structurally distinct anti-inflammatory agents, particularly targeting smoke-induced inflammation and associated pulmonary diseases.

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

Introduction: Osteoarthritis (OA) is a degenerative joint condition characterized by chronic pain and the need for pain management. Locally targeting the endocytotic AP2 complex in nociceptors presents a potential strategy for providing sustained pain relief in individuals with OA. Objective: We investigated whether pain behavior associated with OA can be mitigated by genetically silencing the AP2alpha2 subunit of the AP2 complex in nociceptors and by pharmacologically inhibiting the AP2 complex through the intraarticular administration of a small lipidated decoy peptide. Method: Monoiodoacetate (MIA) was employed to induce knee joint OA in mice and rats. Pain behavior was assessed using dynamic weight-bearing and von Frey filaments. Upon confirmation of established OA pain behavior, in vivo AP2alpha2 genetic knockdown in mice was achieved through sciatic nerve transfection of a targeting AP2alpha2 short hairpin RNA (shRNA). To pharmacologically target endocytosis, a single intraarticular injection of peptide was administered into the arthritic knee of rats. The injection contained either the AP2 inhibitor peptide or a scrambled peptide control. Results: Pain behavior was significantly reduced after both genetic and pharmacological disruption of AP2-driven endocytosis. Animals treated with the Ap2 inhibitor peptide exhibited reduced pain behavior throughout the 28-day assay period. Following the completion of behavioral testing, arthritic knee joints and contralateral healthy knee joints were subsequently collected to assess the impact of the treatment on disease progression. Micro-computed tomography analysis revealed a preservation of bone volume in the arthritic joints that received the AP2 inhibitor peptide treatment, in contrast to the scrambled peptide group. Conclusion: These findings demonstrate that the inhibition of nociceptor endocytosis by a small lipidated peptide presents a promising approach to provide sustained relief from joint pain in individuals with arthritis.

BackgroundAnesthetic agents administered during surgery are one of the key perioperative factors affecting immune modulation in cancer patients. This comparative study elucidated the mechanisms by which the volatile anesthetic, isoflurane and the intravenous anesthetic, propofol impacted apoptosis signaling in CD4+ helper T (Th) cells. MethodsFlow cytometry was used to analyze apoptosis, mitochondrial function and reactive oxygen species (ROS) generation, while Western blotting, ELISA and RT-qPCR were employed to study protein/gene expression in sorted CD4{square} Th cells from perioperative breast cancer female patients (anesthetized with isoflurane or propofol, n=15 per group) and Jurkat T cells. ResultsPatient-derived CD4{square} Th cells and Jurkat T cells exhibited that isoflurane at clinically relevant concentrations triggered apoptosis through mitochondrial depolarization, ROS generation, DNA damage, and activation of caspase-3/7. Specific use of caspase-3/7 inhibitor, Z-DEVD-FMK and antioxidant N-acetyl cysteine rescued isoflurane-induced apoptosis. Further, isoflurane relative to propofol, activated p38 mitogen-activated protein kinase (MAPK), and use of p38 inhibitor, SB203580 suppressed isoflurane-induced apoptosis. Collectively, these findings validated the involvement of the ROS-p38-caspase-3/7 axis in isoflurane-associated apoptosis signaling. On the other hand, propofol conserved mitochondrial integrity, reduced oxidative stress, and maintained higher proliferative capacity. Interestingly, isoflurane-associated apoptosis was transient, with postoperative recovery in patients and similar rescue from apoptosis was evident in Jurkat T cells within 24-48 h of drug removal. ConclusionsBy integrating analyses of patient-derived CD4+ Th cells with mechanistic validations in Jurkat T cells, this study identified the ROS-p38-caspase-3/7 signaling axis and the reversible nature of isoflurane-induced apoptosis.

Background Treatment of Raynaud phenomenon(RP) with oral vasodilators(calcium channel block-ers and phosphodiesterase type 5 inhibitors) has shown moderate efficacy, may not benefit to all patients, and adverse effects often compromise long-term treatment. In addition, a large placebo effect may jeopardize the assessment of treatment benefits. Pharmaconutritional strategies aiming at increasing nitric oxide bioavailability (beet-root juice and L-citrulline) may be promising alternatives, and we further hypothesized that patient preference for a treatment could be a driver of the response. Methods This study consisted of a series of randomized, double-blind, N-of-1 trials conducted in outpa-tients with primary or secondary RP. Each patient underwent a multiple crossover design with repeated blocks of randomized treatments periods: 2 weeks of placebo, 2 weeks of active treat-ments, and 1 week of washout. Outcomes included the Raynaud Condition Score(RCS), fre-quency and daily duration of attacks. Each patient prespecified its preferred primary outcome, efficacy threshold and preferred treatment, which was used for stratified randomization. Gener-alized linear mixed-effects models were used to determine individual and aggregated efficacy. Results Twenty-one patients completed 2 to 8 treatment blocks. Seventeen patients tested L-citrulline, 17 beetroot juice and 13 both treatments. Ten patients selected RCS as a primary outcome, 6 patients the number of attacks and 5 the duration of attacks. Me-dian threshold for considering treatment efficacy chosen by patients was 50% (min-max 20% to 75%) reduction of symptoms. Using individual criteria to define efficacy neither L-citrulline nor beetroot juice showed significant efficacy compared to baseline. Based on the aggregated data, our results show no significant difference between L-citrulline and the L-citrulline-based placebo, nor between beetroot juice and nitrate-depleted beetroot juice, with the exception of the daily duration of RP attacks with beetroot juice (p=0.002). Finally, there was a marked placebo response, notably when patients received their preferred treatment. Conclusions: Our study did not show significant beetroot juice or L-citrulline efficacy in RP. However, we found that individual preference for one treatment over another maximizes responses to both placebo and active treatments, particularly with regard to the frequency and duration of RP attacks, thus suggesting that a real and modifiable placebo effect exists in RP.

Breast cancer (BC) is the second most common noncutaneous cancer and the second leading cause of cancer-related death in women. BC is classified into three primary subtypes, with triple-negative breast cancer (TNBC) having the poorest prognosis because it lacks specific targetable markers. Preclinical studies on TNBC indicated a common occurrence of diminished tumor-suppressor activity of PTEN, activating the PI3K/AKT/mTOR signaling pathway. Notably, published studies reveal that the WWP1 enzyme plays a pivotal role in driving PTEN degradation via ubiquitination, unveiling a promising therapeutic target for treating TNBC. In the search of new WWP1 inhibitors, we used artificial intelligence (AI)-driven computational strategies for de novo design of peptide-based WWP1 inhibitors and identified a hexapeptide, termed WI23-B, which demonstrated high nanomolar binding affinity to WWP1. In TR-FRET enzymatic assays, WI23-B inhibited WWP1 activity with an IC of approximately 11 {micro}M. In MCF7 and MDA-MB-231 breast cancer cell lines, WI23-B showed promising cytotoxic efficacy, particularly in combination with the PI3K inhibitor BYL719, also when it was loaded into nanocapsules. Collectively, these findings highlight WI23-B as a promising lead peptide with potent WWP1 inhibitory activity and synergistic antiproliferative effects when combined with PI3K inhibitors. While further structural optimization is required to enhance its potency and pharmacological properties, our results provide a strong foundation for the development of next-generation WWP1 inhibitors. Such agents have the potential to reshape therapeutic strategies for BC and TNBC by enabling more effective and less toxic treatment regimens, ultimately reducing the reliance on high-dose chemotherapy and minimizing adverse effects.

Background: Complementary and Alternative Medicine (CAM) therapies, such as massage, meditation, and yoga, are widely used to promote wellness, including sleep improvement. Although some CAM therapies may improve sleep through stress reduction, relaxation, and relief of physical discomfort, little is known about associations between individual CAM modalities and sleep health at the population level. Therefore, we investigated the associations between CAM therapies and short sleep duration as well as insomnia symptoms. Methods: Participants from the nationally-representative 2012 National Health Interview Survey (NHIS) self-reported the use of CAM therapies and short sleep duration (<7 hours vs. 7-9 hours) as well as insomnia symptoms (yes vs. no). Poisson regression with robust variance was used to estimate adjusted prevalence ratios (aPRs) and 95% confidence intervals (CIs) for cross-sectional associations between CAM therapy use and sleep outcomes. Results: Among 30,405 participants, the average age was 46.1 +/- 0.2 years and 51% were women. Adults reporting any vs. no CAM therapy use had a higher prevalence of short sleep duration (aPR: 1.11; 95% CI: 1.05-1.16) and insomnia symptoms (aPR: 1.56; 95% CI: 1.47-1.65) after adjustment for sociodemographic and clinical characteristics. Herbal supplements (aPR: 1.13; 95% CI: 1.07-1.19) and massage (aPR: 1.14; 95% CI: 1.05-1.23) were associated with higher prevalence of short sleep duration. Most CAM therapies were associated with higher prevalence of insomnia symptoms, with the strongest associations observed for meditation/guided imagery/progressive relaxation (aPR: 1.84; 95% CI: 1.68-2.02). Conclusion: The higher prevalence of short sleep duration and insomnia symptoms among CAM users may reflect reverse causation, as adults with more severe or persistent sleep disturbances may be more likely to seek CAM therapies. Longitudinal studies are needed to clarify directionality.